Céline Aguer

Glutathionylation acts as a control switch for uncoupling proteins UCP2 and UCP3

The mitochondrial uncoupling proteins 2 and 3 (UCP2 and -3) are known to curtail oxidative stress and participate in a wide array of cellular functions, including insulin secretion and the regulation of satiety. However, the molecular control mechanism(s) governing these proteins remains elusive. Here we reveal that UCP2 and UCP3 contain reactive cysteine residues that can be conjugated to glutathione. We further demonstrate that this modification controls UCP2 and UCP3 function. Both reactive oxygen species and glutathionylation were found to activate and deactivate UCP3-dependent increases in non-phosphorylating respiration. We identified both Cys25 and Cys259 as the major glutathionylation sites on UCP3. Additional experiments in thymocytes from wild-type and UCP2 null mice demonstrated that glutathionylation similarly diminishes non-phosphorylating respiration. Our results illustrate that UCP2- and UCP3-mediated state 4 respiration is controlled by reversible glutathionylation. Altogether, these findings advance our understanding of the roles UCP2 and UCP3 play in modulating metabolic efficiency, cell signaling, and oxidative stress processes.

Galactose Enhances Oxidative Metabolism and Reveals Mitochondrial Dysfunction in Human Primary Muscle Cells

Background Human primary myotubes are highly glycolytic when cultured in high glucose medium rendering it difficult to study mitochondrial dysfunction. Galactose is known to enhance mitochondrial metabolism and could be an excellent model to study mitochondrial dysfunction in human primary myotubes. The aim of the present study was to 1) characterize the effect of differentiating healthy human myoblasts in galactose on oxidative metabolism and 2) determine whether galactose can pinpoint a mitochondrial malfunction in post-diabetic myotubes. Methodology/Principal Findings Oxygen consumption rate (OCR), lactate levels, mitochondrial content, citrate synthase and cytochrome C oxidase activities, and AMPK phosphorylation were determined in healthy myotubes differentiated in different sources/concentrations of carbohydrates: 25 mM glucose (high glucose (HG)), 5 mM glucose (low glucose (LG)) or 10 mM galactose (GAL). Effect of carbohydrates on OCR was also determined in myotubes derived from post-diabetic patients and matched obese non-diabetic subjects. OCR was significantly increased whereas anaerobic glycolysis was significantly decreased in GAL myotubes compared to LG or HG myotubes. This increased OCR in GAL myotubes occurred in conjunction with increased cytochrome C oxidase activity and expression, as well as increased AMPK phosphorylation. OCR of post-diabetic myotubes was not different than that of obese non-diabetic myotubes when differentiated in LG or HG. However, whereas GAL increased OCR in obese non-diabetic myotubes, it did not affect OCR in post-diabetic myotubes, leading to a significant difference in OCR between groups. The lack of an increase in OCR in post-diabetic myotubes differentiated in GAL was in relation with unaltered cytochrome C oxidase activity levels or AMPK phosphorylation. Conclusions/Significance Our results indicate that differentiating human primary myoblasts in GAL enhances aerobic metabolism. Because this cell culture model elicited an abnormal response in cells from post-diabetic patients, it may be useful in further studies of the molecular mechanisms of mitochondrial dysfunction.

Acylcarnitines: potential implications for skeletal muscle insulin resistance

Insulin resistance may be linked to incomplete fatty acid β‐oxidation and the subsequent increase in acylcarnitine species in different tissues including skeletal muscle. It is not known if acylcarnitines participate in muscle insulin resistance or simply reflect dysregulated metabolism. The aims of this study were to determine whether acylcarnitines can elicit muscle insulin resistance and to better understand the link between incomplete muscle fatty acid β‐oxidation, oxidative stress, inflammation, and insulin‐resistance development. Differentiated C2C12, primary mouse, and human myotubes were treated with acylcarnitines (C4:0, C14:0, C16:0) or with palmitate with or without carnitine acyltransferase inhibition by mildronate. Treatment with C4:0, C14:0, and C16:0 acylcarnitines resulted in 20–30% decrease in insulin response at the level of Akt phosphorylation and/or glucose uptake. Mildronate reversed palmitate‐induced insulin resistance concomitant with an ~25% decrease in short‐chain acylcarnitine and acetylcarnitine secretion. Although proinflammatory cytokines were not affected under these conditions, oxidative stress was increased by 2–3 times by short‐ or long‐chain acylcarnitines. Acylcarnitine‐induced oxidative stress and insulin resistance were reversed by treatment with antioxidants. Results are consistent with the conclusion that incomplete muscle fatty acid β‐oxidation causes acylcarnitine accumulation and associated oxidative stress, raising the possibility that these metabolites play a role in muscle insulin resistance.—Aguer, C., McCoin, C. S., Knotts, T. A., Thrush, A. B., Ono‐Moore, K., McPherson, R., Dent, R., Hwang, D. H., Adams, S. H., Harper, M.‐E. Acylcarnitines: potential implications for skeletal muscle insulin resistance. FASEB J. 29, 336–345 (2015). www.fasebj.org

Muscle uncoupling protein 3 overexpression mimics endurance training and reduces circulating biomarkers of incomplete β-oxidation

Exercise substantially improves metabolic health, making the elicited mechanisms important targets for novel therapeutic strategies. Uncoupling protein 3 (UCP3) is a mitochondrial inner membrane protein highly selectively expressed in skeletal muscle. Here we report that moderate UCP3 overexpression (roughly 3‐fold) in muscles of UCP3 transgenic (UCP3Tg) mice acts as an exercise mimetic in many ways. UCP3 overexpression increased spontaneous activity (~40%) and energy expenditure (~5–10%) and decreased oxidative stress (~ 15–20%), similar to exercise training in wild‐type (WT) mice. The increase in complete fatty acid oxidation (FAO; ~30% for WT and ~70% for UCP3 Tg) and energy expenditure (~8% for WT and 15% for UCP3 Tg) in response to endurance training was higher in UCP3 Tg than in WT mice, showing an additive effect of UCP3 and endurance training on these two parameters. Moreover, increases in circulating short‐chain acylcarnitines in response to acute exercise in untrained WT mice were absent with training or in UCP3 Tg mice. UCP3 overexpression had the same effect as training in decreasing long‐chain acylcarnitines. Outcomes coincided with a reduction in muscle carnitine acetyltransferase activity that catalyzes the formation of acylcarnitines. Overall, results are consistent with the conclusions that circulating acylcarnitines could be used as a marker of incomplete muscle FAO and that UCP3 is a potential target for the treatment of prevalent metabolic diseases in which muscle FAO is affected.—Aguer, C., Fiehn, O., Seifert, E. L., Bézaire, V., Meissen, J. K., Daniels, A., Scott, K., Renaud, J.‐M., Padilla, M., Bickel, D. R., Dysart, M., Adams, S. H., Harper, M.‐E. Muscle uncoupling protein 3 overexpression mimics endurance training and reduces circulating biomarkers of incomplete β‐oxidation. FASEB J. 27, 4213–4225 (2013). www.fasebj.org

Hexokinase II acts through UCP3 to suppress mitochondrial reactive oxygen species production and maintain aerobic respiration

UCP3 (uncoupling protein-3) mitigates mitochondrial ROS (reactive oxygen species) production, but the mechanisms are poorly understood. Previous studies have also examined UCP3 effects, including decreased ROS production, during metabolic states when fatty acid oxidation is high (e.g. a fasting state). However, the role of UCP3 when carbohydrate oxidation is high (e.g. fed state) has remained largely unexplored. In the present study, we show that mitochondrial-bound HK (hexokinase) II curtails oxidative stress and enhances aerobic metabolism of glucose in the fed state in a UCP3-dependent manner. Genetic knockout or inhibition of UCP3 significantly decreased mitochondrial-bound HKII. Furthermore, UCP3 was required for the HKII-mediated decrease in mitochondrial ROS emission. Intriguingly, the UCP3-mediated modulation of mitochondria-associated HKII was only observed in cells cultured under high-glucose conditions. UCP3 was required to maintain high rates of aerobic metabolism in high-glucose-treated cells and in muscle of fed mice. Deficiency in UCP3 resulted in a metabolic shift that favoured anaerobic glycolytic metabolism, increased glucose uptake and increased sensitivity to oxidative challenge. PET (positron emission tomography) of [18F]fluoro-deoxyglucose uptake confirmed these findings in UCP3-knockout and wild-type mice. Collectively, our findings link the anti-oxidative and metabolic functions of UCP3 through a surprising molecular connection with mitochondrial-bound HKII.

Increased FAT/CD36 Cycling and Lipid Accumulation in Myotubes Derived from Obese Type 2 Diabetic Patients

Background Permanent fatty acid translocase (FAT/)CD36 relocation has previously been shown to be related to abnormal lipid accumulation in the skeletal muscle of type 2 diabetic patients, however mechanisms responsible for the regulation of FAT/CD36 expression and localization are not well characterized in human skeletal muscle. Methodology/Principal Findings Primary muscle cells derived from obese type 2 diabetic patients (OBT2D) and from healthy subjects (Control) were used to examine the regulation of FAT/CD36. We showed that compared to Control myotubes, FAT/CD36 was continuously cycling between intracellular compartments and the cell surface in OBT2D myotubes, independently of lipid raft association, leading to increased cell surface FAT/CD36 localization and lipid accumulation. Moreover, we showed that FAT/CD36 cycling and lipid accumulation were specific to myotubes and were not observed in reserve cells. However, in Control myotubes, the induction of FAT/CD36 membrane translocation by the activation of (AMP)-activated protein kinase (AMPK) pathway did not increase lipid accumulation. This result can be explained by the fact that pharmacological activation of AMPK leads to increased mitochondrial beta-oxidation in Control cells. Conclusion/Significance Lipid accumulation in myotubes derived from obese type 2 diabetic patients arises from abnormal FAT/CD36 cycling while lipid accumulation in Control cells results from an equilibrium between lipid uptake and oxidation. As such, inhibiting FAT/CD36 cycling in the skeletal muscle of obese type 2 diabetic patients should be sufficient to diminish lipid accumulation.

Intrinsic aerobic capacity correlates with greater inherent mitochondrial oxidative and H2O2 emission capacities without major shifts in myosin heavy chain isoform.

Exercise capacity and performance strongly associate with metabolic and biophysical characteristics of skeletal muscle, factors that also relate to overall disease risk. Despite its importance, the exact mechanistic features that connect aerobic metabolism with health status are unknown. To explore this, we applied artificial selection of rats for intrinsic (i.e., untrained) aerobic treadmill running to generate strains of low- and high-capacity runners (LCR and HCR, respectively), subsequently shown to diverge for disease risk. Concurrent breeding of LCR and HCR per generation allows the lines to serve as reciprocal controls for unknown environmental changes. Here we provide the first direct evidence in mitochondria isolated from skeletal muscle that intrinsic mitochondrial capacity is higher in HCR rats. Maximal phosphorylating respiration was ~40% greater in HCR mitochondria, independent of substrate and without altered proton leak or major changes in protein levels or muscle fiber type, consistent with altered control of phosphorylating respiration. Unexpectedly, H(2)O(2) emission was ~20% higher in HCR mitochondria, due to greater reduction of more harmful reactive oxygen species to H(2)O(2); indeed, oxidative modification of mitochondrial proteins was lower. When the higher mitochondrial yield was considered, phosphorylating respiration and H(2)O(2) emission were 70-80% greater in HCR muscle. Greater capacity of HCR muscle for work and H(2)O(2) signaling may result in enhanced and more immediate cellular repair, possibly explaining lowered disease risks.

Four‐week cold acclimation in adult humans shifts uncoupling thermogenesis from skeletal muscles to brown adipose tissue

Muscle‐derived thermogenesis during acute cold exposure in humans consists of a combination of cold‐induced increases in skeletal muscle proton leak and shivering. Daily cold exposure results in an increase in brown adipose tissue oxidative capacity coupled with a decrease in the cold‐induced skeletal muscle proton leak and shivering intensity. Improved coupling between electromyography‐determined muscle activity and whole‐body heat production following cold acclimation suggests a maintenance of ATPase‐dependent thermogenesis and decrease in skeletal muscle ATPase independent thermogenesis. Although daily cold exposure did not change the fibre composition of the vastus lateralis, the fibre composition was a strong predictor of the shivering pattern evoked during acute cold exposure.

Abnormal metabolism flexibility in response to high palmitate concentrations in myotubes derived from obese type 2 diabetic patients

Insulin resistance in type 2 diabetes (T2D) is associated with intramuscular lipid (IMCL) accumulation. To determine whether impaired lipid oxidation is involved in IMCL accumulation, we measured expression of genes involved in mitochondrial oxidative metabolism or biogenesis, mitochondrial content and palmitate beta-oxidation before and after palmitate overload (600μM for 16h), in myotubes derived from healthy subjects and obese T2D patients. Mitochondrial gene expression, content and network were not different between groups. Basal palmitate beta-oxidation was not affected in T2D myotubes, whereas after 16h of palmitate pre-treatment, T2D myotubes in contrast to control myotubes, showed an inability to increase palmitate beta-oxidation (p<0.05). Interestingly, acetyl-CoA carboxylase (ACC) phosphorylation was increased with a tendency for statistical significance after palmitate pre-treatment in control myotubes (p=0.06) but not in T2D myotubes which can explain their inability to increase palmitate beta-oxidation after palmitate overload. To determine whether the activation of the AMP activated protein kinase (AMPK)-ACC pathway was able to decrease lipid content in T2D myotubes, cells were treated with AICAR and metformin. These AMPK activators had no effect on ACC and AMPK phosphorylation in T2D myotubes as well as on lipid content, whereas AICAR, but not metformin, increased AMPK phosphorylation in control myotubes. Interestingly, metformin treatment and mitochondrial inhibition by antimycin induced increased lipid content in control myotubes. We conclude that T2D myotubes display an impaired capacity to respond to metabolic stimuli.

Four-week cold acclimation in adult humans shifts uncoupling thermogenesis from skeletal muscles to BAT

Muscle‐derived thermogenesis during acute cold exposure in humans consists of a combination of cold‐induced increases in skeletal muscle proton leak and shivering. Daily cold exposure results in an increase in brown adipose tissue oxidative capacity coupled with a decrease in the cold‐induced skeletal muscle proton leak and shivering intensity. Improved coupling between electromyography‐determined muscle activity and whole‐body heat production following cold acclimation suggests a maintenance of ATPase‐dependent thermogenesis and decrease in skeletal muscle ATPase independent thermogenesis. Although daily cold exposure did not change the fibre composition of the vastus lateralis, the fibre composition was a strong predictor of the shivering pattern evoked during acute cold exposure.